The objectives of our research are to elucidate how enzymes catalyze chemical reactions, and to utilize this information to discover specific inhibitors of potential chemotherapeutic utility. We focus on the mechanisms of enzymes which are involved in purine nucleoside and nucleotide metabolism; these are essential processes in all organisms. In one project we are investigating the synthesis and utilization of S-adenosylmethionine (variously abbreviated as AdoMet, SAM, SAM-e), which is essential to the life of all cellular organisms. S-adenosylmethionine participates in a multitude of pathways that regulate gene expression and cell growth, processes of substantial importance in carcinogenesis. We are investigating some of the varied types of chemical reactions in which AdoMet participates, reactions that range from alkylation to free radical formation. We also study structure-function relationships in inosine monophosphate dehydrogenase (IMPDH), which catalyzes a critical reaction in guanine nucleotide biosynthesis. IMPDH is an established target for anti-cancer and immunosuppressive drugs. A combination of kinetic, spectroscopic and computational techniques are used to correlate enzyme structure and function. Other studies use computational methods to probe the structures and reactivity of organoboron compounds; boron containing compounds are increasingly used in drugs and in diagnostic reagents. Our objective is to understand the complex chemical bonding to boron and to develop computationally efficient ways to model these structures.Top

Pimkin

Inosine-5'-monophosphate dehydrogenase (IMPDH, IMP:NAD oxidoreductase), catalyzes the rate limiting step in guanine nucleotide biosynthesis, the oxidation of IMP to xanthosine monophosphate (XMP). IMPDH has a central role in DNA and RNA syntheses, in G-protein mediated signal transduction, as well as in intermediary metabolism. IMPDH is an established target for anti-cancer, immunosuppressive, anti-viral and anti-microbial chemotherapies. In addition to the catalytic core of the protein, IMPDH has a ca. 120-residue subdomain that is appended as a loop in the protein structure (Figure: Structure of the IMPDH subunit); the subdomain is conserved in IMPDH throughout evolution, and has sequence homology to a segment of cystathionine β-synthase and numerous other proteins of unrelated function. The in vivo importance of this IMPDH subdomain is clear since human retinitis pigmentosa RP10 is caused by sequence variants in this domain. To probe the role of this domain in a model system, we constructed an E. coli strain in which the subdomain of the chromosomal gene has been precisely excised, with preservation of the both the core sequence and IMPDH catalytic function. The physiological effects of this deletion are being determined by quantitative proteomic and metabolic analyses. The results indicate a role for the subdomain in regulation of intracellular nucleotide metabolism.Top

Lu, Hussein

S-adenosylmethionine decarboxylase (Ado-MetDC) is a pyruvoyl cofactor-dependent enzyme that participates in polyamine biosynthesis. The polyamines have essential roles in cell growth, and drugs that prevent their synthesis have been used in cancer and antimicrobial therapies. The E. coli enzyme is a prototype of a microbial group of AdoMet decarboxylases, which require a metal ion activator, in contrast to their eukaryotic counterpart. The E. coli AdoMetDC has an usual metal ion selectivity; ions with charges of +1, +2 and +3 are able to activate, and all provide similar maximal catalytic rates, suggesting that they do not influence the rate-limiting catalytic step. The free energy of cation binding is directly related to the charge/ionic-radius ratio of the ion, a startlingly simple relationship previously unseen in metal-protein interactions. The binding of metal ion activators and enzyme activation are both cooperative with Hill coefficients as large as 4, indicating that the binding of one cation can modify the entire (αβ) ₄ enzyme, suggesting action by a profound protein conformational alteration. Spectroscopic experiments are being used to locate the metal ion binding site within the protein, for which the structure is unknown. The results are consistent with allosteric metal ion activation of the enzyme, congruent with the role of the organic cation putrescine activator of the mammalian AdoMet decarboxylase. Top

Taylor

S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, a.k.a., methionine adenosyltransferase, MAT) catalyzes a unique and metabolically essential reaction. The multitude of metabolic roles of AdoMet, its involvement in the modulation of cell growth, and the change of the synthetase isoform present in mammalian liver tumors, has led to a decades long search for inhibitors with in vitro and in vivo utility. We have continued a computational virtual screen in which more than 1,000,000 compounds have been evaluated for potential affinity to the crystallographically defined active site of the enzyme. Several structurally unrelated candidate compounds have been evaluated by in vitro enzyme assays, as well as tested for inhibition of bacterial, yeast, and mammalian cell growth. Inhibitors with submicromolar affinity that are structurally unrelated to the natural ligands have been identified. In each case the substrate ATP prevents inhibition, substantiating the proposed binding at the active site. These compounds provide leads towards inhibitors with enhanced bioavailability.Top

McVicker

Structural genomics projects are providing large numbers of three-dimensional structures of proteins of unknown function (POUFs). In conjunction with the improvements of computer algorithms for in silico docking of small molecules to protein structures that have been driven by the drug discovery field, an opportunity has appeared for guiding the elucidation of the metabolic roles of these POUFs via docking of three-dimensional structures of metabolites to the protein structures. Remarkably, a suitable library of metabolite structures is not available. In order to enable the discovery process, we continue to construct a three-dimensional structural library containing several thousands of primary and secondary metabolites. These structures have correct bonding, stereochemistry, physiologically relevant ionization states, and energy-minimized three-dimensional structures.Top

Bock, in collaboration with Larkin, Schaeffer, Bhat, Lai

Organoboronic acids are important pharmaceutical agents which act as selective transporters of nucleosides and saccharides, as inhibitors of proteases, and as cancer therapeutic agents in boron neutron capture therapy. Boronic acids (R-B(OH) 2) react with compounds containing 1,2- or 1,3-diols to form pharmacologically relevant cyclic boronate esters. Nevertheless, many factors that influence these reactions are not well understood, particularly the ability of amines to catalyze ester formation. We are using density functional theoretical methods to examine mechanisms such as that in which a primary aliphatic amine acts as an internal Lewis base to catalyze the formation of a boron-oxygen-carbon (B-O-C) linkage in the methanolysis of H₂N-CH₂-CH=CH-B(OH)₂ to produce H₂N-CH₂-CH=CH-B(OH)(OCH₃). The catalytic role of the primary amine group during the methanolysisof H₂N-CH-sub>2-CH=CH-B(OH)₂ results from facilitation of a proton transfer from an intermolecular B-O dative-bonded adduct between methanol and this boronic acid, rather than having the anticipated origin in an intramolecular B-N dative bond.Top